Cancer Cell Biology
Lack of functional erythropoietin receptors of cancer cell lines
Article first published online: 7 NOV 2007
Copyright © 2007 Wiley-Liss, Inc.
International Journal of Cancer
Volume 122, Issue 5, pages 1005–1011, 1 March 2008
How to Cite
Laugsch, M., Metzen, E., Svensson, T., Depping, R. and Jelkmann, W. (2008), Lack of functional erythropoietin receptors of cancer cell lines. Int. J. Cancer, 122: 1005–1011. doi: 10.1002/ijc.23201
- Issue published online: 24 DEC 2007
- Article first published online: 7 NOV 2007
- Manuscript Accepted: 18 JUL 2007
- Manuscript Received: 23 FEB 2007
- Ministry of Science, Economy and Commerce of Schleswig-Holstein. Grant Number: BA 410
- erythropoietin receptor;
- tumor growth;
Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation. © 2007 Wiley-Liss, Inc.
The human erythropoietin receptor (EpoR) is a transmembrane protein of 484 amino acids and a calculated mass of 52.6 kDa,1 which increases to about 60 kDa due to glycosylation and phosphorylation. When Epo binds 2 cell-surface EpoR molecules, EpoR-associated Janus tyrosine kinase-2 (JAK2) are activated.2 The intracellular signaling pathways involve STAT5, Ras/mitogen-associated protein kinase (Ras/MAPK) and phosphatidylinositol-3 kinase/Akt1 (PI-3K/Akt1). EpoR is also expressed by nonhematopoietic tissues, including the cardiovascular and the central nervous systems.3
Treatment of cancer patients with recombinant human erythropoiesis stimulating agents (rhESA) reduces transfusion requirements and improves quality of life.4–6 Anemia prevention is important with a view to hypoxia-driven tumor progression.7 However, the negative outcomes of high-dose rhESA therapy trials on patients with breast8 or head and neck cancers9 have raised concern that Epo may promote tumor growth. A prerequisite for effects of Epo is the existence of functional EpoR. Previous studies have provided conflicting results,5, 10 which may be partly due to nonspecificity of the antibodies used for detection of EpoR protein.11
Herein, we tested a panel of cancer cell lines for expression of EpoR mRNA and EpoR protein. EpoR was identified by EpoR knockdown in cells transfected with EpoR cDNA and small interfering RNA (siRNA). Epo induced phosphorylation of JAK2, STAT5 or other molecules and growth stimulation only in Epo-depending human UT7/Epo leukemia cells, but not in the cancer cell lines tested.
Material and methods
Cell culture conditions
Cells of the human lines SH-SY5Y (neuroblastoma), HepG2 (hepatoma), HeLa (cervix carcinoma), MCF7 (breast carcinoma), U2-OS (osteosarcoma), HEK293T (embryonic kidney), renal carcinoma cell 4 (RCC4; VHL-deficient renal clear cell carcinoma), HCT116 and p53HCT116 (colon carcinoma), 7860wt and SW480 (colon carcinoma), K562 (erythroleukemia) and the CHO line (Chinese hamster ovary) were maintained in RPMI 1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS; Invitrogen/Gibco, Karlsruhe, Germany), penicillin and streptomycin in a humidified atmosphere containing 5% CO2 in air (normoxia). The EpoR expressing human leukemia cell line UT7/Epo12 was grown in DMEM/F12 supplemented with 1 U Epo/mL (Epoetin beta; Roche/Boehringer, Mannheim, Germany). Experiments were carried out in DMEM with 0.5% FCS or in OptiMEM (Invitrogen/Gibco). Hypoxic incubation was performed in a humidified incubator (Ruskinn Technology, Leeds, UK) with 1 or 3% O2, 5% CO2 and balance N2 for 24 or 48 hr.
EpoR encoding DNA from the plasmid hEpoRpMOWS (kindly provided by Dr. Ursula Klingmüller; DKFZ, Heidelberg, Germany) was subcloned into pcDNA3. U2-OS, HEK293T, HeLa, HepG2 and CHO cells were transiently transfected with hEpoRpcDNA3 and/or siRNA (10 and 20 pmol/μL) for EpoR knockdown with Lipofectamine 2000 (Invitrogen/Gibco).
Western blot analysis and immunohistochemistry
Cells were washed 3 times with PBS and harvested in lysis buffer containing 0.5% NP-40 and protease inhibitor cocktail (Calbiochem, Darmstadt, Germany). After incubation at 4°C for 1 hr the lysates were centrifuged at 14,000g and 4°C for 10 min. Optionally, extracts were subjected to immunoprecipitation (1 mL lysate, 50 μL protein A-agarose beads; Amersham Pharmacia Biotech, Freiburg, Germany) with the antibodies: M20 (sc-697; polyclonal anti-murine EpoR; Santa Cruz Biotechnology, Heidelberg, Germany), C20 (sc-695; polyclonal anti-human EpoR; Santa Cruz Biotechnology), MAB307 (monoclonal anti-human EpoR; R&D Systems, Wiesbaden, Germany) or 07-311 (polyclonal anti-murine EpoR; Upstate, Charlottesville, VA). For enrichment of glycoproteins, 1 mL of supernatants was incubated with Agarose Wheat Germ Lectin (WGA) or Concanavalin A (ConA) Sepharose 4B (Amersham Pharmacia Biotech) at 4°C for 60 min. On immunoprecipitation with C20 antibody, proteins were identified by MALDI-MS analysis of trypsin digested proteins of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) protein spots (MALDI-MS spectra are available upon request; WITA, Tetlow, Germany). Whole cell lysates or precipitates were incubated in SDS-sample buffer under reducing conditions at 95°C for 5 min. After SDS-PAGE (8% PA), proteins were electrotransferred onto membranes (Hybond, Amersham) that were blocked with 5% skim milk in PBS. Membranes were incubated at 4°C overnight with 1 of the following antibodies diluted in PBS: M20 (1:500), C20 (1:1,000) or MAB307 (1:1,000). When noted, C20-antibody was preincubated (1:1) with blocking peptide for EpoR (C-terminal 20 amino acids of human EpoR, C20p; Santa Cruz Biotechnology). To confirm cellular hypoxia, blots were performed with the monoclonal anti-human hypoxia-inducible factor 1α (HIF-1α) antibody (1:1,000 in PBS; BD Transduction Laboratories, Heidelberg, Germany) as described.13 Immunoblots with anti-α-tubulin (1:1,000 in PBS; Santa Cruz Biotechnology) served as loading controls. Secondary antibodies (goat anti-rabbit or goat anti-mouse; DakoCytomation, Glostrup, Denmark) were conjugated with horseradish peroxidase. Immunoreactive bands were detected with enhanced chemiluminescence (Amersham).
For immunofluorescence, cells cultured on poly-L-lysine coated coverslips overnight were fixed with 10% paraformaldehyde for 10 min, incubated with 0.5% Triton X-100 in PBS for 5 min and with 5% skim milk for 1 hr. Anti-EpoR antibodies were diluted 1:1,000 in 5% skim milk/PBS. The secondary antibody was labeled with Alexa™594 and diluted 1:1,000 in PBS. The cells were permanently mounted in Mowiol 4-88 (Calbiochem) for fluorescence microscopy (Axioplan 2000; Carl Zeiss, Oberkochen, Germany).
EpoR mRNA quantification
RNA was isolated with the ABI Prism™ 6100 Nucleic Prepstation (Applied Biosystems, Darmstadt, Germany). cDNA was generated by Superscript™ III RNase H-reverse transcriptase (Invitrogen/Gibco). EpoR cDNA was quantified in duplicate on an ABI 7000 Sequence detection system by means of a SYBR green PCR kit (Applied Biosystems) and program: 50°C for 2 min, 95°C for 10 min, 95°C for 12 sec, 60°C for 1 min for 40 cycles. Primer sequences for EpoR were forward 5′-CAAGTTCGAGAGCAAAGCGG-3′ (exon 1) and reverse 5′-TTCCTCCCAGAAACACACCAAG-3′ (exon 2). To demonstrate response to hypoxia14 glucose transporter 1 (Glut1) cDNA was quantified (Assays on demand; Applied Biosystems).15 For normalization of cDNA data, 60S ribosomal protein L28 gene product was quantified (1:5,000 dilution). Primer sequences for L28 were forward 5′-ATGGTCGTGCGGAACTGCT-3′ and reverse 5′-TTGTAGCGGAAGGAATTGCG-3′. All oligonucleotides were from Invitrogen/Gibco. Expression levels were calculated with the ΔΔcT method and related to the respective normoxic control cultures. Specificity of the EpoR primer was verified by agarose gel electrophoresis and melting point curve analysis. In addition, representative RT-PCR products from 6 cell lines were subjected to sequence analysis (SeqLab, Göttingen, Germany).
Cells in 12-well dishes were washed 3 times with PBS and maintained in OptiMEM overnight and then treated with Epo (0, 0.01, 0.1, 1 and 10 U/mL) for 5 min. The medium was removed and 100 μL boiling SDS 3× sample buffer was added. Following 10% SDS-PAGE and blotting, the membranes were incubated with phosphospecific polyclonal anti-JAK2 and anti-Akt1 or monoclonal phosphospecific anti-STAT5 and anti-Erk1/2 (New England Biolabs, Frankfurt, Germany) and antiphosphotyrosine (BioSource, Camarillo, CA) or phosphorylation-independent rabbit polyclonal anti-Erk2 antibodies (Santa Cruz Biotechnology).
Effects of Epo on cell growth were studied by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich, Taufkirchen, Germany) assay in 96-well microtiter plates as reported.16
EpoR mRNA was expressed by all cell lines tested (U2-OS, HepG2, HeLa, HCT116, p53-HCT116 and SW480; Fig. 1a). The level of EpoR cDNA was not affected by hypoxia exposure (24 hr) while Glut1 mRNA expression was stimulated (Fig. 1b). Neither EpoR mRNA nor Glut1 mRNA expression increased on Epo treatment (shown for HepG2 cells in Fig. 1c).
All antibodies reacted with (Fig. 2) and immunoprecipitated (not shown) more than 1 protein. MALDI-MS sequence analysis of proteins precipitated with C20 from U2-OS cell-lysates failed to show EpoR, but identified several proteins, 1 of which was the100-kDa heat-shock protein HSP90b. The mass spectra and fingerprinting analysis of this protein spot resulted in a sequence coverage of 21% of the HSP90b protein sequence and the following 12 peptides were identified: (i) IDIIPNPQER, (ii) TLTLVDTGIGMTK, (iii) ADLINNLGTIAK, (iv) ADHGEPIGR, (v) VILHLKEDQTEYLEER, (vi) TKPIWTR, (vii) HFSVEGQLEFR, (viii) RAPFDLFENK, (ix) GVVDSEDLPLNISR, (x) YHTSQSGDEMTSLSEYVSR, (xi) EQVANSAFVER, (xii) EGLELPEDEEEKK.
None of the bands was strengthened on hypoxia or Epo treatment (Figs. 2 and 3a). The 60-kDa EpoR protein produced after transfection of U2-OS cells with hEpoRpcDNA3 had the same size as the endogenous EpoR protein of UT7/Epo cells. Only this band disappeared after transfection with siRNA preventing EpoR formation (Fig. 2d). The 60-kDa protein was prominent, when N-glycosylated proteins were purified by WGA or α-D-mannopyranosyl, and α-D-glucopyranosyl and sterically related residues were purified by ConA from UT7/Epo cell lysates (Fig. 3c).
On immunohistochemistry, K562 cells showed mainly plasma membrane staining with C20 antibody, whereas U2-OS and RCC4 cells displayed cytoplasmic fluorescence. The 07-311 antibody stained the nuclei of RCC4 cells (data not shown). HEK293 cells showed fluorescence in plasma membranes when incubated with MAB307, but cytoplasmatic staining with M20 and, more strongly, with C20 antibody (data not shown). HEK293T cells transfected with hEpoRpcDNA3 yielded small clusters after staining with M20 or C20, which increased on Epo treatment (Fig. 4).
The question has been raised whether cancer cells possess functional EpoR. We detected EpoR mRNA in all tumor cell lines examined, which is consistent with earlier studies.17, 18 EpoR mRNA levels were unaffected by Epo application or hypoxia exposure for 24 hr. The possibility still remains that hypoxia causes a transient activation of the EpoR gene, since EpoR mRNA levels have been reported to increase in SiHa cervical19 and MCF7 breast cancer cells20, 21 on hypoxia exposure for short periods (3–4 hr). Hypoxia-response elements containing HIF-1 binding sites in control of the EpoR gene have not been characterized, although hypoxia activates the EpoR gene in endothelial cells.22
There are several reports of EpoR protein detection in human tumor tissues subjected to immunohistochemistry or Western blotting.19, 20, 23–29 In all of these studies, the C20 antibody raised against the 20 C-terminal amino acids of human EpoR was used for staining. In our immunohistochemical studies, C20 antibody, a monoclonal anti-human antibody MAB 307 and two polyclonal anti-murine antibodies (M20, 07-311) yielded staining of different cell compartments (membrane, cytosol, nucleus). Although in erythrocytic cells most of the newly synthesized EpoR molecules are rapidly degraded to 46- and 39-kDa fragments,30 the differences in localization and size of stained proteins from native versus EpoR cDNA transfected cells indicate that posttranslational modifications of EpoR were not responsible for the variability in staining. A more likely explanation is cross-reaction with proteins not related to EpoR, which renders these antibodies unsuitable for immunohistochemical or immunofluorescent demonstration of EpoR.
Of primary interest is the most commonly used C20 antibody. Apparently, some proteins share amino acid sequence similarity with the C-terminus of EpoR. When we transiently knocked down the EpoR gene in U2-OS cells by siRNA transfection, only the 60-kDa protein was lost. Thus, the other proteins that were stained were neither generated by alternative splicing, which can occur in cancer cells,31 nor by posttranslational modification of EpoR. In fact, C20 antibody stains renal cancer 769-P cells that lack EpoR.11 With the same antibody Elliott et al.11 stained a 66-kDa protein, which they identified as HSP70. Brown et al.32 have recently confirmed this finding and reported that preincubation of C20 antibody with either HSP70-2 or HSP70-5 causes abolition of cytoplasmic immunoreactivity in nonsmall cell lung carcinoma tissue. By sequence analysis of C20-immunoprecipitates from U2-OS cultures we identified the 100-kDa band as HSP90. Likewise, M20 is a frequently used antibody for EpoR immunoblots. This antibody interacts with various proteins in crude lysates. Most of these are not glycoproteins, whereas EpoR is a glycoprotein. Enrichment of glycosylated proteins from crude extracts by WGA or ConA treatment led to a reduced number of proteins detected and a strong 60-kDa band, which is the predicted size of EpoR. ConA and WGA fractionation provide glycoproteins which are typical for the cis and trans Golgi subcompartments.33
Epo strongly induced phosphorylation of JAK2, STAT5 and other tyrosine containing proteins in UT7/Epo cells. Recently, Dunlop et al.34 have demonstrated that EPO treatment can activate JAK2/STAT5, Ras/ERK and PI-3K pathways in nonsmall cell lung carcinoma cell lines. Of note is our failure to detect JAK2 activation in any of the cancer cell lines because JAK2 phosphorylation is an initiating event of EpoR signaling and, consequently, essential for STAT5, Ras/MAPK and PI-3K activation.35 Moreover, JAK2 is required for trafficking of newly synthesized EpoR from endoplasmic reticulum into the cell membrane.36 The lack of STAT5 activation is noteworthy, because STAT5, through binding to the bcl-x promoter, is a major transcriptional factor to inhibit apoptosis.37 Indeed, when tested over a wide concentration range, Epo neither altered viability nor growth rate, as assessed by MTT-assay, in the present cancer cell lines. This result is in line with several reports,17, 38–43 though not with all.19, 44, 45 The reason for these discrepant findings is not clear, but one should be aware that minute amounts of Epo stabilizing additives may affect cell viability when protein-free culture medium is used. However, we cannot exclude the possibility that Epo has an effect on cancer cells that depends on other growth factors missing in our experimental setup.
Studies of the combined effects of Epo and chemotherapeutics on cancer cells in culture have not provided conclusive results. One group has reported that RCCs undergo a higher degree of apoptosis with a combination of daunorubicin or vinblastine and Epo than with either of these agents alone.46 In contrast to this chemosensitization Epo was found to cause resistance to cis-platin in HeLa19 and to dacarbazine in melanoma cells.29 In the latter studies, extremely high Epo concentrations were necessary, which were about 1,000-fold higher than the plasma concentration reached in cancer patients under rhESA therapy.
An explanation is required for the cytoprotective and proliferative effects occasionally seen by investigators applying high doses of Epo in cancer cell cultures. Depending on the cell line and culture conditions, Epo may act via EpoR/βcR rather than via the homodimeric EpoR. Tissue protection by Epo in nonhemopoietic tissues is thought to be mediated by EpoR covalently complexed with βcR.47, 48 One has to consider that a recent study showed cytoprotection by Epo48 in SH-SY5Y and PC-12 neuronal cells, although βcR was not detectable.49 An approach to test whether high-dose treatment of cancer cells with Epo results in EpoR/βcR signaling in cancer cells would be to transfect cells with siRNA to inhibit βcR formation. We were unable to perform these experiments because cancer cells did not respond to Epo in the first place.
The study was supported by a grant from the Ministry of Science, Economy and Commerce of Schleswig-Holstein (M. Laugsch; HWP, BA 410). W.J. has served as consultant and received hornoraria from Amgen, Roche, Shire and Ortho Biotech. The other authors declare to have no potential conflict of interest.
- 5Cancer patient survival and erythropoietin. J Natl Compr Canc Netw 2005; 3: 796–804..